It has recently been shown that specific proteins in mammalian cells are modified by the covalent attachment of prenyl groups to a cysteine residue at the C-terminus via a thioether linkage. Some proteins, such as lamin B, contain a 15-carbon farnesyl group. The majority of the prenylated proteins, including the gamma-subunit of brain G-proteins, contain a 20-carbon geranylgeranyl group. We plan to continue our studies in the area of protein prenylation by focusing on three areas. Since my laboratory discovered the geranylgeranylation of proteins, a logical next step will be to purify and characterize the enzyme(s) involved in transferring the prenyl group to the cysteine residue. The substrate specificity of the prenyl transferase will be examined in order to determine the specificity rules for the geranylgeranylation versus farnesylation of proteins. The prenylation occurs on a cysteine residue that is four amino acids in from the C-terminus of the immature proteins. The last three amino acids are removed so that the prenylated cysteine is now the C-terminal residue. We plan to purify and characterize the protease(s) involved in the C-terminal hydrolysis of prenylated proteins. We also plan to develop a general synthetic strategy for the preparation of lipidated peptides. We plan to synthesize a C-terminal peptide derived from human ras proteins that is both farnesylated and palmitoylated and to use this peptide o study the membrane-binding properties to synthetic phopholipid vesicles. Both the membrane affinity and intermembrane exchanges rate will be determined by using fluorescence resonance energy transfer. This synthetic peptide will also be radio-iodinated for use in the search for specific cell- membranes receptors that bind to prenylated proteins. Similar studies will be carried out with lipidated peptides derived from farnesylated, but non-palmitoylated, forms of ras proteins and from the gamma-subunit of G-proteins. Finally, we will prepare analogs of the yeast a-factor. This phermone has been recently shown to contain a farnesyl group and we will explore the role of the prenyl group in the recognition by the yeast a-factor receptor.